G Protein β1γ2 Subunits Promote Microtubule Assembly

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108

112

G proteins serve many functions involving the transfer of signals from cell surface receptors to intracellular effector molecules. Considerable evidence suggests that there is an interaction between G proteins and the cytoskeleton. In this report, G protein ␣ subunits G i1 ␣, G s ␣, and G o ␣ are shown to activate the GTPase activity of tubulin, inhibit microtubule assembly, and accelerate microtubule dynamics. G i ␣ inhibited polymerization of tubulin-GTP into microtubules by 80 -90% in the absence of exogenous GTP. Addition of exogenous GTP, but not guanylylimidodiphosphate, which is resistant to hydrolysis, overcame the inhibition. Analysis of the dynamics of individual microtubules by video microscopy demonstrated that G i1 ␣ increases the catastrophe frequency, the frequency of transition from growth to shortening. Thus, G␣ may play a role in modulating microtubule dynamic instability, providing a mechanism for the modification of the cytoskeleton by extracellular signals.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 91%

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G Protein α Subunits Activate Tubulin GTPase and Modulate Microtubule Polymerization Dynamics

Roychowdhury

Panda

Wilson

et al. 1999

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108

112

“…Each of these scenarios are also of note relative to the role of AGS3 (PINS)-G␣ i / G␣ o complexes in neuroblast processing in D. melanogaster and the defect in orientation of the mitotic spindle observed in the absence of PINS (9,10). In the latter situation, interaction of AGS3 (PINS) with G␣ i(GDP) /G␣ o(GDP) could "release" G␤␥ for effector regulation, which may involve the apparent localization of G␤␥ to microtubules and/or mitotic spindles (24,25). The detailed analysis of the interaction of AGS3 with G␣ presented in this report should greatly facilitate efforts to further FIG.…”

Section: Discussionmentioning

confidence: 99%

Selective Interaction of AGS3 with G-proteins and the Influence of AGS3 on the Activation State of G-proteins

Bernard

Peterson

Chung

et al. 2001

142

196

AGS3 (activator of G-protein signaling 3) was isolated in a yeast-based functional screen for receptor-independent activators of heterotrimeric G-proteins. As an initial approach to define the role of AGS3 in mammalian signal processing, we defined the AGS3 subdomains involved in G-protein interaction, its selectivity for G-proteins, and its influence on the activation state of Gprotein. Immunoblot analysis with AGS3 antisera indicated expression in rat brain, the neuronal-like cell lines PC12 and NG108-15, as well as the smooth muscle cell line DDT 1 -MF2. Immunofluorescence studies and confocal imaging indicated that AGS3 was predominantly cytoplasmic and enriched in microdomains of the cell. AGS3 coimmunoprecipitated with G␣ i3 from cell and tissue lysates, indicating that a subpopulation of AGS3 and G␣ i exist as a complex in the cell. The coimmunoprecipitation of AGS3 and G␣ i was dependent upon the conformation of G␣ i3 (GDP > > GTP␥S (guanosine 5-3-O-(thio)triphosphate)). The regions of AGS3 that bound G␣ i were localized to four amino acid repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus (Pro 463 -Ser 650 ), each of which were capable of binding G␣ i . AGS3-GPR domains selectively interacted with G␣ i in tissue and cell lysates and with purified G␣ i /G␣ t . Subsequent experiments with purified G␣ i2 and G␣ i3 indicated that the carboxyl-terminal region containing the four GPR motifs actually bound more than one G␣ i subunit at the same time. The AGS3-GPR domains effectively competed with G␤␥ for binding to G␣ t(GDP) and blocked GTP␥S binding to G␣ i1 . AGS3 and related proteins provide unexpected mechanisms for coordination of G-protein signaling pathways.Signal processing via heterotrimeric G-protein proteins generally involves an initial input sensed by a cell surface receptor with seven membrane-spanning regions. Conformational changes in receptor subdomains then transfer this signal to a G-protein, promoting exchange of GTP for GDP and subunit dissociation with both the G␣ and G␤␥ subunits regulating effector molecules. These events are tightly regulated to maximize signal efficiency, optimize signal specificity, and integrate cellular responses to diverse stimuli. Regulatory mechanisms include the segregation of specific signaling molecules in cell microdomains, receptor phosphorylation and internalization, cross-talk between signaling pathways, and proteins that regulate the basal activation state of G-proteins independently of the receptor.We partially purified a direct G-protein activator from NG108-15 cells (1, 2) and subsequently used a functional screen to identify three proteins (AGS1-3, for activator of Gprotein signaling 1-3) that activated heterotrimeric G-protein signaling in the absence of a cell surface receptor (3-5). The identification of such proteins raises many interesting and unexpected questions relative to signal processing by heterotrimeric G-proteins. As an initial approach to address these issues, we focused on the biochemical and functional characterizati...

“…Due to their interaction with transmembrane receptors and lipid modification, G proteins are usually associated with the plasma membrane. Recently, increasing evidence has emerged that G proteins are also present at intracellular areas such as Golgi apparatus, endoplasmic reticulum, cytoskeleton, and even the nucleus (1)(2)(3)(4)(5). Moreover, some studies have also shown that activated G␣s can be released from the plasma membrane to the cytoplasm (6 -9).…”

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confidence: 99%

A Specific Domain of Giα Required for the Transactivation of Giα by Tubulin Is Implicated in the Organization of Cellular Microtubules

Chen

Skiba

et al. 2003

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␣ subunits bind tubulin with high affinity, whereas transducin (G t ␣) does not. The interaction between tubulin and G␣, which also involves the direct transfer of GTP from tubulin to G␣ (transactivation), is not yet fully understood. This study, using chimeras of G i ␣ and G t ␣, showed that the G i ␣ (215-295) segment converted G t ␣ to bind to tubulin and this chimera (chimera 1) could be transactivated by tubulin. Insertion of G t ␣ (237-270) into chimera 1 to form chimera 2 resulted in a protein that, like G t ␣, did not bind tubulin. Thus, it was thought that the G i ␣ (237-270) domain was essential to modulate the binding of G i ␣ 1 to tubulin. Surprisingly, when domain (237-270) of G i ␣ was replaced by G t ␣ (237-270) to form chimera 3, the chimera bound to tubulin with a similar affinity (K D Х120 nM) as wild-type G i ␣ 1 . However, even though chimera 3 displayed normal GTP binding, it was not transactivated by GTP-tubulin. Furthermore, when these chimeras were expressed in COS-1 cells, cellular processes in cells overexpressing G i ␣ 1 or chimera 1 were more abundant and longer than those in native cells. G␣ was seen throughout the length of the process. Morphology of cells expressing chimera 2 was identical to controls. Consistent with the role of Chimera 3 as a "dominant negative" G␣, cells transfected with chimera 3 had only few truncated processes. This study demonstrates that although G i ␣ (237-270) is not obligatory for the binding of G i ␣ to tubulin, it is crucial for the transactivation of G␣ by tubulin. These results also suggest that the transactivation of G␣ by tubulin may play an important role in modulating microtubule organization and cell morphology.G proteins act as intracellular transducers to propagate a variety of signals across the plasma membrane. Due to their interaction with transmembrane receptors and lipid modification, G proteins are usually associated with the plasma membrane. Recently, increasing evidence has emerged that G proteins are also present at intracellular areas such as Golgi apparatus, endoplasmic reticulum, cytoskeleton, and even the nucleus (1-5). Moreover, some studies have also shown that activated G␣s can be released from the plasma membrane to the cytoplasm (6 -9). Thus, it is possible that G proteins exist in several different cellular locations and play roles in various physiologic processes.Microtubules, a major component of the cytoskeleton, are involved in many cellular functions including chromosome movements during mitosis, intracellular transport, and the modulations of cell morphology. The biological function of microtubules is based, in significant part, on the ability of tubulin to polymerize and depolymerize. A heterodimer of ␣-and ␤-tubulin is the basic building block of microtubules. Tubulin is a GTP-binding protein, two GTP molecules are bound noncovalently in exchangeable (E-site on ␤-tubulin) and nonexchangeable (N-site on ␣-tubulin) sites. Both G␣ subunits and tubulin have intrinsic GTPase activity but that of tubulin is activated durin...